Bacterial infections caused by staphylococcus bacteria (i.e., a “staph infection”) are very common in the general population. About 25% of individuals commonly carry staphylococcus bacteria on their skin or in their nose. Most of the time, these bacteria cause no problem or result in relatively minor skin infections. However, staph infections can turn deadly if the bacteria invade deeper into an individual's body, for example, entering the bloodstream, joints, bones, lungs or heart. In the past, a lethal staph infection might have occurred in a person who was hospitalized or had a chronic illness or weakened immune system. Now, it is increasingly common for an otherwise healthy individual to develop life-threatening staph infections. Importantly, many staph infections no longer respond to common antibiotics.
Staphylococcus aureus, often referred to as “staph,” Staph. aureus,” or “S. aureus,” is a major human pathogen, producing a multitude of virulence factors making it able to cause several types of infection, from superficial lesions to toxinoses and life-threatening systemic conditions such as endocarditis, osteomyelitis, pneumonia, meningitis and sepsis (reviewed in Miller and Cho, “Immunity Against Staphylococcus aureus Cutaneous Infections,” Nat. Rev. Immunol. 11:505-518 (2011)). Although most individuals encounter S. aureus shortly after birth (Holtfreter et al., “Towards the Immune Proteome of Staphylococcus aureus—The Anti-S. aureus Antibody Response,” Int. J. Med. Microbiol. 300:176-192 (2010)) and possess both antibodies against S. aureus and the ability to increase anti-S. aureus titers after infection, these antibodies are often not protective against recurrent S. aureus infections (Foster T J, “Immune Evasion by Staphylococci,” Nat. Rev. Microbiol. 3:948-958 (2005)). In the United States alone, an annual mortality of more than 20,000 is attributed to methicillin-resistant S. aureus (MRSA), exceeding deaths caused by influenza, viral hepatitis, and HIV/AIDS (Foster, T J., “Immune Evasion by Staphylococci,” Nat. Rev. Microbiol. 3:948-958 (2005); Klevens et al., “The Impact of Antimicrobial-Resistant, Health Care-Associated Infections on Mortality in the United States,” Clin. Infect. Dis. 47:927-930 (2008)). The pathogen produces a variety of molecules that presumably facilitate survival in or on the human host.
Bi-component, pore-forming leukotoxins are among the secreted virulence factors produced by S. aureus. These toxins can be secreted as water soluble monomers which oligomerize, and then insert pores into the plasma membrane, which subsequently disrupt the cellular osmotic balance and membrane potential leading to death of the targeted cells, most notably polymorphonuclear leukocytes (PMNs) and mononuclear phagocytes (Bischogberger et al., “Pathogenic Pore-Forming Proteins: Function and Host Response,” Cell Host Microbe 12(3):266-275 (2012), which is hereby incorporated by reference in its entirety). In the case of Leukotoxin ED (LukED), the targeting, binding, and killing of host phagocytic cells occurs via the cellular target CCR5, CXCR1 and CXCR2 located on the surface of the phagocytes (Alonzo III et al., “Staphylococcus aureus Leucocidin ED Contributes to Systemic Infection by Targeting Neutrophils and Promoting Bacterial Growth In Vivo,” Mol. Microbiol. 83:423-435 (2012); Alonzo III et al. “CCR5 is a Receptor for Staphylococcus aureus Leukotoxin ED,” Nature 493(7430)51-55 (2012); and Reyes-Robles et al., “Staphylococcus aureus Leukotoxin ED Targets the Chemokine Receptors CXCR1 and CXCR2 to Kill Leukocytes and Promote Infection,” Cell Host & Microbe 14:453-459 (2013)). Indeed, when the cellular target of LukED, CCR5, is not present on host immune cells, the host animal is resistant to the otherwise lethal S. aureus infection (Alonzo III et al. “CCR5 is a Receptor for Staphylococcus aureus Leukotoxin ED,” Nature 493(7430):51-55 (2012)). Leukotoxin AB (LukAB) can also kill host phagocytic cells, and its cytolytic activity can be exerted both from the outside and from the inside of the cell, after the microorganism is phagocytosed into the host cell (Dumont et al., “Staphylococcus aureus LukAB Cytotoxin Kills Human Neutrophils by Targeting the CD11b Subunit of the Integrin Mac-1,” PNAS 110(26):10794-10799 (2013)). Due to the contribution that both of these leukotoxins have to pathogenesis, they have been considered critical S. aureus virulence factors (Alonzo III and Tones, “Bacterial Survival Amidst an Immune Onslaught: The Contribution of the Staphylococcus aureus Leukotoxins,” PLOS Path 9(2):e1003143 (2013)).
Another critical factor for the pathogenic success of S. aureus depends on the properties of its surface proteins (Clarke et al., “Surface Adhesins of Staphylococcus aureus,” Adv. Microb. Physiol. 51:187-224 (2006); Patti et al., “MSCRAMM-Mediated Adherence of Microorganisms to Host Tissues,” Annu. Rev. Microbiol. 48:585-617 (1994); and Patti et al., “Microbial Adhesins Recognizing Extracellular Matrix Macromolecules,” Curr. Opin. Cell Biol. 6:752-758 (1994)).
S. aureus employs microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) in order to adhere to and colonize host tissues. MSCRAMMs can recognize collagen, heparin-related polysaccharides, fibrinogen, and/or fibronectin. S. aureus expresses a subset of MSCRAMMs, which all contain the serine-aspartate dipeptide repeat (SDR) domain, including clumping factor A (ClfA), clumping factor B (ClfB), SdrC, SdrD, and SdrE (Becherelli et al. “Protective Activity of the CnaBE3 Domain Conserved Among Staphylococcus aureus Sdr Proteins,” PLoS One 8(9): e74718 (2013)). S. epidermidis also expresses three members of this family, SdrF, SdrG, and SdrH (McCrea et al., “The Serine-Aspartate Repeat (Sdr) Protein Family in Staphylococcus Epidermidis,” Microbiology 146:1535-1546 (2000)). All of these proteins share a similar structure comprising an N-terminal ligand-binding A domain followed by the SDR domain, which can contain between 25-275 serine-aspartate dipeptide repeats. The C-terminal portion of these proteins contains the LPXTG-motif, which facilitates cell wall anchoring by the transpeptidase sortase A. The serine-aspartate dipeptide regions in SDR-containing proteins are modified by the sequential addition of glycans by two glycosyltransferases. First, SdgB appends N-acetylglucosamine (GlcNAc) on serine residues within the serine-aspartate dipeptide regions, followed by SdgA modification of the glycoprotein, resulting in disaccharide moieties. This glycosylation can protect SDR-containing staphylococcal proteins from Cathepsin G-mediated degradation (Hazenbos et al., “Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins,” PLoS Pathog 9(10):e1003653 (2013)).
Additionally, Protein A, also located on the surface of S. aureus, contributes to staphylococcal escape from protective immune responses by capturing the Fc domain of host IgG, as well as the Fab domain of the VH3 clan of IgG and IgM (Sjodahl et al., “Repetitive Sequences in Protein A from Staphylococcus aureus. Arrangement of Five Regions Within the Protein, Four Being Highly Homologous and Fc-Binding,” Eur. J. Biochem. 73:343-351 (1997); and Cary et al., “The Murine Clan V(H) III Related 7183, J606 and S107 and DNA4 Families Commonly Encode for Binding to a Bacterial B cell Superantigen,” Mol. Immunol. 36:769-776 (1999)).
Additionally, S. aureus expresses a second immunoglobulin binding protein referred to as the second binding protein for immunoglobulins (Sbi) (Zhang et al., “A Second IgG-Binding Protein in Staphylococcus aureus,” Microbiology 144:985-991 (1998) and Atkins et al., “S. aureus IgG-binding Proteins SpA and Sbi: Host Specificity and mechanisms of Immune Complex Formation,” Mol. Immunol. 45:1600-1611 (2008)) that is either secreted or associated with the cell envelope (Smith et al., “The Sbi Protein is a Multifunctional Immune Evasion Factor of Staphylococcus aureus” Infection & Immunity 79:3801-3809 (2011) and Smith et al., “The Immune Evasion Protein Sbi of Staphylococcus aureus Occurs both Extracellularly and Anchored to the Cell Envelope by Binding to Lipotechoic Acid” Mol. Microbiol. 83:789-804 (2012)) and shares a pair of conserved helices with Protein A involved in binding to the Fc region of IgG proteins (Atkins et al., “S. aureus IgG-binding Proteins SpA and Sbi: Host Specificity and mechanisms of Immune Complex Formation,” Mol. Immunol. 45:1600-1611 (2008)).
Furthermore, S. aureus secretes a number of proteases that have been implicated in immune evasion. Rooijakkers et al. demonstrated that S. aureus secretion of staphylokinase, a plasminogen activator protein, led to the activation of plasmin that cleaved both surface-bound IgG and complement C3b, ultimately reducing immune-mediated S. aureus destruction (Rooijakkers et al., “Anti-Opsonic Properties of Staphylokinase,” Microbes and Infection 7:476-484 (2005)). S. aureus also secretes the serine protease glutamyl endopeptidase V8 (GluV8) that can directly cleave human IgG1 in the lower hinge region between E233 and L234 (EU numbering (Edelman et al., “The Covalent Structure of an Entire GammaG Immunoglobulin Molecule,” PNAS 63:78-85 (1969), Brerski et al., “Human Anti-IgG1 Hinge Autoantibodies Reconstitute the Effector Functions of Proteolytically Inactivated IgGs,” J. Immunol. 181:3183-3192 (2008)). It was also recently demonstrated that human anti-S. aureus IgGs are rapidly cleaved when bound to the surface of S. aureus (Fernandez Falcon et al., “Protease Inhibitors Decrease IgG Shedding From Staphylococcus aureus, Increasing Complement Activation and Phagocytosis Efficiency,” J. Med. Microbiol. 60:1415-1422 (2011)).
Taken together, these studies indicate that S. aureus utilizes a number of mechanisms that could adversely affect standard IgG1-based monoclonal antibody (mAb) therapeutics, either by directly cleaving the mAb, sequestering the mAb by Protein A binding, or by killing off the very effector cells required for therapeutic efficacy. It is therefore not surprising that presently there are no mAb-based therapies targeting S. aureus that have achieved final approval for use in humans. Thus, there remains a need for methods and compositions that can treat staphylococcal infection, which (i) evade protein A and Sbi binding, (ii) escape staph-induced proteolysis, (iii) can neutralize leukotoxins and (iv) are capable of opsonizing and delivering S. aureus to phagocytes. The present application meets these and other needs.
Summary
The present disclosure provides multi-specific binding molecules comprising at least a first binding domain and a second binding domain, each of which specifically binds to a different bacterial virulence factor in particular a different staphylococcal virulence factor. The first and the second binding domains are optionally connected via a linker peptide.
The first binding domain is capable of binding to a glycosylated staphylococcal surface protein, such as an staphylococcal SDR-containing protein. In one aspect, the staphylococcal SDR-containing protein is ClfA, ClfB, SdrC, SdrD, SdrE, SdrF, SdrG and SdrH. Preferably, the staphylococcal SDR-containing protein is ClfA, ClfB, SdrC, SdrD or SdrE.
In one aspect, the first binding domain is a full-length antibody or antibody fragment. Preferably, the full-length antibody or antibody fragment is resistant to proteolytic degradation by a staphylococcal protease that cleaves wild-type IgG1 (such as a staphylococcal protease, e.g., Staphylococcus aureus V8 protease, that cleaves wild-type IgG1 between or at residues 222-237 (EU numbering) within SEQ ID NO:60). In another aspect, the full-length antibody or antibody fragment is a human, humanized, or chimeric antibody or antibody fragment.
In one aspect, the binding molecule is not capable of specific binding to human FcγRI, is not capable of specific binding to Protein A, and is not capable of specific binding to Sbi. In one aspect, the binding molecule is capable of specific binding to FcRn.
In one aspect, the first binding domain comprises an immunoglobulin heavy chain variable (VH) region having the amino acid sequence selected from the group of VH region amino acid sequences of SEQ ID NOs:60, 62, 64 or 66. In another aspect, the first binding domain comprises an immunoglobulin light chain variable (VL) region having the amino acid sequence of SEQ ID NOs:61, 63, 65 or 67. Alternatively, the first binding domain comprises (a) a VH region having the amino acid sequence of SEQ ID NOs:60, 62, 64 or 66; and (b) a VL region having the amino acid sequence of SEQ ID NOs:61, 63, 65 or 67. In one embodiment, the first binding domain comprises (1) a VH region having the amino acid sequence of SEQ ID NO:60, and a VL region having the amino acid sequence of SEQ ID NO:61; (2) a VH region having the amino acid sequence of SEQ ID NO:62, and a VL region having the amino acid sequence of SEQ ID NO:63; (3) a VH region having the amino acid sequence of SEQ ID NO:64, and a VL region having the amino acid sequence of SEQ ID NO:65; (4) a VH region having the amino acid sequence of SEQ ID NO:66, and a VL region having the amino acid sequence of SEQ ID NO:67; (5) a VH region having the amino acid sequence of SEQ ID NO:68, and a VL region having the amino acid sequence of SEQ ID NO:69; (6) a VH region having the amino acid sequence of SEQ ID NO:70, and a VL region having the amino acid sequence of SEQ ID NO:71; (7) a VH region having the amino acid sequence of SEQ ID NO:72, and a VL region having the amino acid sequence of SEQ ID NO:73; (8) a VH region having the amino acid sequence of SEQ ID NO:74, and a VL region having the amino acid sequence of SEQ ID NO:75; (9) a VH region having the amino acid sequence of SEQ ID NO:76, and a VL region having the amino acid sequence of SEQ ID NO:77; or (10) a VH region having the amino acid sequence of SEQ ID NO:78, and a VL region having the amino acid sequence of SEQ ID NO:79.
In yet another aspect, the binding molecule comprises (a) a first binding domain that comprises (i) a VH region having the amino acid sequence of SEQ ID NOs:60, 62, 64 or 66; and (ii) a VL region having the amino acid sequence of SEQ ID NOs:61, 63, 65 or 67; and (b) a second binding domain that comprises the amino acid sequence of any one of SEQ ID NOs:14-59 or SEQ ID NOs:113-666. In once aspect, the second binding domain comprises the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:22.
The second binding domain is capable of binding to a staphylococcal leukotoxin. In one aspect, the staphylococcal leukotoxin is selected from a group consisting of leukotoxin A (LukA), leukotoxin B (LukB), leukotoxin AB (LukAB), leukotoxin D (LukD), leukotoxin E (LukE), leukotoxin ED (LukED), Panton-Valentine leukocidin S (LukS-PV), Panton-Valentine leukocidin F (LukF-PV), Panton-Valentine leukocidin (LukSF/PVL), gamma hemolysin A (HlgA), gamma hemolysin C (HlgC), gamma hemolysin B (HlgB), gamma hemolysin AB (HlgAB), and gamma-hemolysin BC (HlgBC). In one aspect, the staphylococcal leukotoxin is LukAB, LukD, or LukE.
In one aspect, the second binding domain is an alternative scaffold. In one aspect, the alternative scaffold comprises a fibronectin type III (FN3) domain. In one aspect, the FN3 domain binds to LukA having the amino acid sequence of SEQ ID NO:10, LukB having the amino acid sequence of SEQ ID NO:11, LukD having the amino acid sequence of SEQ ID NO:12, or LukE having the amino acid sequence of SEQ ID NO:13. Alternatively, the FN3 domain binds to a LukAB complex comprising LukA having the amino acid sequence of SEQ ID NO:10 and LukB having the amino acid sequence of SEQ ID NO:11, and/or a LukED complex comprising LukE having the amino acid sequence of SEQ ID NO:13 and LukD having the amino acid sequence of SEQ ID NO:12.
In one aspect, the second binding domain of the binding molecule competes with an FN3 domain having an amino acid sequence of any one of SEQ ID NOs:14-59 or SEQ ID NOs:113-666 for binding to (i) LukA having the amino acid sequence of SEQ ID NO:10; (ii) LukB having the amino acid sequence of SEQ ID NO:11; (iii) LukD having the amino acid sequence of SEQ ID NO:12; and/or (iv) LukE having the amino acid sequence of SEQ ID NO:13.
In one aspect, the second binding domain is a FN3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:14-59 and SEQ ID NOs:113-666. In one aspect, the second binding domain is a FN3 domain comprising the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:22.
In one aspect, the binding molecule further comprises one or more additional binding domains, where the one or more additional binding domains are capable of (i) binding to a different glycosylated staphylococcal surface protein than bound by the first binding domain and/or (ii) binding to a different staphylococcal leukotoxin than bound by the second binding domain.
The disclosure also provides nucleic acid sequences encoding the above described binding molecules as well as vectors comprising a nucleic acid sequence or nucleic acid sequences. Additionally, the invention contemplates a host cell comprising these vectors.
Moreover, the disclosure provides a process for the production of the above described binding molecules, comprising (a) culturing a host cell containing a vector comprising a nucleic acid sequence or nucleic acid sequences that encode the inventive binding molecules, and (b) recovering the binding molecule from the culture.
Furthermore, the disclosure provides a pharmaceutical composition comprising a binding molecule as described herein.
The binding molecules provided herein can be used in the treatment, prevention or amelioration of a staphylococcal infection. In one aspect, the staphylococcal infection is caused by Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA). Accordingly, the present disclosure provides a method for the treatment, prevention or amelioration of a staphylococcal infection, comprising (a) administering to a subject in need thereof a binding molecule as described herein. This method of treatment, prevention of amelioration of staphylococcal infection may involve repeated administration of the binding molecule as described herein, or administration of the binding molecule in combination with one or more other agents, such as an anti-infective agent, an antibiotic agent, and/or an antimicrobial agent.
The binding molecules provided herein can also be used in methods for diagnosing a staphylococcal infection in a subject. In one aspect, the method for diagnosing a staphylococcal infection involves contacting the binding molecule as described herein with a sample from a subject and detecting the presence or the absence of a glycosylated staphylococcal surface protein and/or a staphylococcal leukotoxin in the sample based on the contacting. This method further involves diagnosing the staphylococcal infection in the subject based on the detection of the glycosylated staphylococcal surface protein and/or the staphylococcal leukotoxin in the sample.
Similar methods can be employed for the detection of a staphylococcus in a sample. In particular, the methods for detection of staphylococcus in a sample involve contacting the binding molecule as described herein with a sample, and detecting the presence or the absence of a glycosylated staphylococcus surface protein and/or a staphylococcal leukotoxin in the sample based on the contacting, whereby the presence of the glycosylated staphylococcus surface protein and/or staphylococcal leukotoxin indicates the presence of staphylococcus in the sample.
Furthermore, the binding molecules of the present invention can be used for the prevention of a staphylococcal infection. In one aspect, a method for the prevention of a staphylococcal infection involves contacting the binding molecule described herein with a sample from the subject, and detecting a glycosylated staphylococcal surface protein and/or a staphylococcal leukotoxin in the sample as a result of the contacting. The method further involves administering an agent suitable for preventing staphylococcal infection to the subject based on the detection of a glycosylated staphylococcal surface protein and/or a staphylococcal leukotoxin in the sample.
Also contemplated by the present disclosure is a kit comprising a binding molecule as provided herein, and a kit comprising a nucleic acid molecule encoding the binding molecules provided, a vector comprising a nucleic acid molecule(s) encoding the binding molecules provided and/or a host cell containing such a vector.